Development of diverse skeletal muscle fiber types is controlled by a complex mechanism of cell-cell interactions and signal transduction events leading to expression of contractile protein genes characteristic of the muscle fiber type. During vertebrate skeletal muscle development, innervation of fetal muscle fibers causes expression of slow myosin heavy chain (MyHC) genes in a distinct subpopulation of muscle fibers and thereby establishes and maintains muscle fiber phenotypic differences. Until recently, there has been no appropriate, easily manipulated model system to investigate the mechanism of innervation-induced fiber type development. The goal of the proposed research is to use a novel muscle fiber - motor neuron co-culture system to elucidate the mechanism of fetal fiber type development. The first aim is to define and characterize the intracellular signaling mechanism that regulates slow MyHC gene expression and muscle fiber type. This will be done by use of muscle fiber motor neuron interactions in a new co-culture system and by use of dominant positive and negative mutations of known members of signaling cascades within skeletal muscle fibers in vitro. Emphasis will be placed on the role of guanine nucleotide binding proteins (G proteins) and other signaling molecules in the protein kinase C (PKC) signal transduction cascade. Studies will also focus on the calcineurin signaling pathway and cross-talk between these two signal transduction cascades. Phosphorylation of myogenic regulatory factors (MRFs) and other specific transcription factors by these signaling cascades and subsequent slow MyHC2 gene expression will be examined by expression of non-phosphorylatable transcription factor mutations. The second aim is to identify the regulatory components involved in extrinsic, nerve-dependent regulation of the slow MyHC2 gene in fetal muscle fibers co-cultured with motor neurons. The slow MyHC2 gene with its promoter has been obtained. Identification of specific cis-elements regulating fiber type identity through mechanisms initiated by innervation will be done by transfection of reporter gene constructs transcriptionally driven by slow MyHC2 gene regulatory sequences. Slow MyHC2 gene regulatory regions will be altered by deletion and mutagenesis. The specific aims of the proposal place emphasis on cell and molecular biological approaches to elucidate the extrinsic innervation-induced regulation of skeletal muscle fiber type within in vitro settings that for the first time truly mimic regulation of muscle fiber type in vivo.